HHS Public Access Author manuscript Author Manuscript
Heart Rhythm. Author manuscript; available in PMC 2017 August 01. Published in final edited form as: Heart Rhythm. 2016 August ; 13(8): 1716–1723. doi:10.1016/j.hrthm.2016.05.009.
Arrhythmogenic Calmodulin Mutations Impede Activation of Small-conductance Calcium-Activated Potassium Current Chih-Chieh Yu, MD1,2, Jum-Suk Ko, MD, PhD1,3, Tomohiko Ai, MD, PhD1,4, Wen-Chin Tsai, MD1,5, Zhenhui Chen, PhD1, Michael Rubart, MD1,6, Matteo Vatta, PhD1,7, Thomas H. Everett, IV, PhD.1, Alfred L. George Jr., MD8, and Peng-Sheng Chen, MD, FHRS1 1Krannert
Author Manuscript
Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University, Indianapolis, IN 2Department
of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
3Division
of Cardiology, Department of internal medicine, Wonkwang University School of Medicine and Hospital, Iksan, Korea 4Department
of Molecular Pathogenesis, Medical Research Institute; the Department of Emergency Medicine, Tokyo Medical and Dental University
5Hualein
Tzu-Chi General Hospital, Taiwan
6Department
of Pediatrics, Riley Heart Research Center, Chicago, IL
7Department
of Medical and Molecular Genetics, Indiana University School of Medicine, Chicago,
Author Manuscript
IL 8Department
of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL
Abstract Background—Apamin sensitive small-conductance Ca2+-activated K+ (SK) channels are gated by intracellular Ca2+ through a constitutive interaction with calmodulin. Objective—We hypothesize that arrhythmogenic human calmodulin mutations impede activation of SK channels.
Author Manuscript
Methods—We studied 5 previously published calmodulin mutations (N54I, N98S, D96V, D130G and F90L). Plasmids encoding either wild type (WT) or mutant calmodulin were transiently transfected into human embryonic kidney (HEK) 293 cells that stably express SK2 channels (SK2 Cells). Whole-cell voltage-clamp recording was used to determine apaminsensitive current (IKAS) densities. We also performed optical mapping studies in normal murine
Corresponding Author: Peng-Sheng Chen, MD, 1800 N. Capitol Ave, Room E475, Indianapolis, IN 46202-1228 Telephone: (317) 274-0909, Fax: (317) 962-0588, [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Disclosures: Dr Peng-Sheng Chen has equity interest in Arrhythmotech, LLC. Our laboratory receives equipment donations from Medtronic, St Jude and Cyberonics Inc. Dr. George receives research grants from Gilead Sciences, Inc. and Merck, Inc.
Yu et al.
Page 2
Author Manuscript
hearts to determine the effects of apamin in hearts with (N=7) or without (N=3) pretreatment with sea anemone toxin (ATX II). Results—SK2 cells transfected with WT calmodulin exhibited IKAS density (in pA/pF) of 33.6 [31.4;36.5] (median and confidence interval 25%-75%), significantly higher than that observed for cells transfected with N54I (17.0 [14.0;27.7], p=0.016), F90L (22.6 [20.3;24.3], p=0.011), D96V (13.0 [10.9;15.8], p=0.003), N98S (13.7 [8.8;20.4], p=0.005) and D130G (17.6 [13.8;24.6], p=0.003). The reduction of SK2 current was not associated with a decrease in membrane protein expression or intracellular distribution of the channel protein. Apamin increased the ventricular APD80 (from 79.6 ms [63.4-93.3] to 121.8 ms [97.9-127.2], p=0.010) in hearts pre-treated with ATX-II but not in control hearts. Conclusion—Human arrhythmogenic calmodulin mutations impede the activation of SK2 channels in HEK 293 cells.
Author Manuscript
Keywords Long QT syndrome; catecholaminergic polymorphic ventricular tachycardia; ion channels; cardiac electrophysiology; arrhythmias
Introduction
Author Manuscript Author Manuscript
Small conductance calcium-activated potassium (SK) channels carry a repolarization current responsible for afterhyperpolarization of neurons in the nervous system and contributes to the modulation of intrinsic excitability, synaptic transmission and long-term changes that affect learning and memory formation.1, 2 SK also contributes significantly to the repolarization of atrial myocytes3, 4 and promotes automaticity and atrioventricular node conduction.5 SK is upregulated in atrial fibrillation, heart failure and myocardial infarction.4, 6-8 While SK current is difficult to detect in normal ventricular myocytes at normal pacing rates,3, 6, 9 it is important for ventricular repolarization during bradycardia and atrioventricular (AV) block.10 Apamin is a specific SK current blocker in both neuronal type and cardiac type ion channels.1, 11 SK current blockade by apamin can cause premature ventricular contraction, QT prolongation and torsades de pointes ventricular arrhythmia in normal rabbit ventricles.10 However, there are no reported cases of SK channel mutations in humans with cardiac arrhythmias. Therefore, the importance of SK current in human arrhythmogenesis remains unclear. Calmodulin mutations were reported to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT; N54I, N98S),12 congenital long QT syndrome (LQTS) with recurrent cardiac arrest (D96V, D130G and F142L)13 and childhood and adolescent onset idiopathic ventricular fibrillation (VF) (F90L).14 Because SK channels are gated by intracellular Ca2+ through a constitutive interaction between the pore-forming subunits and calmodulin,1 it is possible that mutant calmodulin may impede the activation of the SK currents. We used human embryonic kidney (HEK) 293 cells that stably express subtype 2 of SK protein (SK2 cells) to test this hypothesis.
Heart Rhythm. Author manuscript; available in PMC 2017 August 01.
Yu et al.
Page 3
Author Manuscript
Methods The study was approved by the Institutional Biosafety Committee and Institutional Animal Care and Use Committee of the Indiana University and the Methodist Research Institute, Indianapolis, Indiana. Detailed methods are included in an online supplement. Transfection of wild type and mutant calmodulins SK2 cells were transfected with various plasmids. The amount of wild type calmodulin plasmids (WT-CaM/pIRES2-EGFP or WT-CaM/pIRES2-dsRED) or mutant calmodulin plasmids (N54I, F90L, D96V, N98S, D130G/pIRES2-EGFP) transfected was either 1 µg each or 1 µg total in combination (for co-expression experiments). Patch-clamp experiments
Author Manuscript
Whole cell voltage-clamp technique was performed.11 The Tyrode’s (bath) solution contains (in mM) NaCl 140, KCl 5.4, MgCl2 1.2, HEPES 5, NaH2PO4 0.33, CaCl2 1.8 and D-glucose 10 (pH 7.4 adjusted with NaOH). The pipette solution contained (in mM) K-Gluconate 144, MgCl2 1.15, EGTA 1, HEPES 10 and different concentration of CaCl2 (pH 7.2 adjusted with KOH). Free Ca2+ concentration was 1.0 μmol/L for all experiments except when testing calcium sensitivities (0.1 to 10 μM). All experiments were carried out at 37°C. SK2 current density was calculated as the difference of current at 0 mV before and after exposure to apamin (500 nM). Immunofluorescence imaging
Author Manuscript
HEK 293 cells and SK2 cells were seeded and grown on coated glass coverslips. The cells were stained with anti-SK2 rabbit polyclonal antibody, incubated with Alexa Fluor 568 goat anti-rabbit IgG secondary and counterstained with DAPI. The cover glasses were then mounted onto a glass slide and imaged by a Leica TCS SP8 laser scanning confocal microscope. To minimize variability, we plated cells on the same day, introduced plasmids in the same way and immunostained all samples altogether. We used the same imaging conditions and acquired images within 2~3 hours to reduce photobleaching. We then compared cells side by side in the same region in the same sample. Western blot analysis
Author Manuscript
HEK 293 cells and SK2 cells were washed in cold PBS and subsequently lysed and homogenized in 10 mM cold sodium bicarbonate. The membrane fractions were harvested by centrifugation. The protein concentrations were measured with Lowry protein assay. Sixty µg of microsomes were subjected to a SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The blot was probed with a rabbit anti-SK2 polyclonal antibody. Antibody-bound protein bands were visualized with 125I-protein A followed by autoradiography. The membrane was then scanned in a flat-bed scanner at 600 dpi and the staining densities were analyzed using ImageJ 1.49b.
Heart Rhythm. Author manuscript; available in PMC 2017 August 01.
Yu et al.
Page 4
Optical mapping studies
Author Manuscript
We used 10 wild type (WT) mice for optical mapping of membrane potential. The hearts were Langendorff perfused with 37°C oxygenated Tyrode’s solution and stained with dye di-4-ANEPPS. Blebbistatin (10 mM) was added to the Tyrode’s solution. Hearts were illuminated with a solid-state laser. Seven hearts were first treated with 15 nM sea anemone toxin (ATX-II) to prolong QT interval, followed by 100 nM of apamin. The remaining 3 hearts were treated with 100 nM of apamin only. The action potential duration measured to 80% repolarization (APD80) was determined at pacing cycle lengths (PCL) of 200 ms. Statistical analysis
Author Manuscript
Data in text and figures are presented as median and confidence interval [25th percentile; 75th percentile] except the figure of normalized calcium sensitivity curve which is presented as mean ± standard error. Nonparametric tests were used for data analyses. Mann-Whitney U test was conducted to compare SK2 current densities between groups with or without transfected calmodulins. Kruskal-Wallis test was performed to test the existence of a statistically significant difference among mutant calmodulins. Repeated measures analyses of variance (RMANOVA) was used to compare the effects of WT:D96V ratio on current density and APD changes in the optical mapping studies. A p value ≤ 0.05 was considered statistically significant. Statistical analyses were performed using SPSS (IBM, Chicago, IL, USA, version 21).
Results
Author Manuscript
Figure 1A illustrates representative current tracings from an SK2 cell before (trace a) and after (trace b) apamin, respectively, obtained by the descending voltage ramp protocol shown in the inset. Trace b shows the small endogenous current (
Heart Rhythm. Author manuscript; available in PMC 2017 August 01. Published in final edited form as: Heart Rhythm. 2016 August ; 13(8): 1716–1723. doi:10.1016/j.hrthm.2016.05.009.
Arrhythmogenic Calmodulin Mutations Impede Activation of Small-conductance Calcium-Activated Potassium Current Chih-Chieh Yu, MD1,2, Jum-Suk Ko, MD, PhD1,3, Tomohiko Ai, MD, PhD1,4, Wen-Chin Tsai, MD1,5, Zhenhui Chen, PhD1, Michael Rubart, MD1,6, Matteo Vatta, PhD1,7, Thomas H. Everett, IV, PhD.1, Alfred L. George Jr., MD8, and Peng-Sheng Chen, MD, FHRS1 1Krannert
Author Manuscript
Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University, Indianapolis, IN 2Department
of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
3Division
of Cardiology, Department of internal medicine, Wonkwang University School of Medicine and Hospital, Iksan, Korea 4Department
of Molecular Pathogenesis, Medical Research Institute; the Department of Emergency Medicine, Tokyo Medical and Dental University
5Hualein
Tzu-Chi General Hospital, Taiwan
6Department
of Pediatrics, Riley Heart Research Center, Chicago, IL
7Department
of Medical and Molecular Genetics, Indiana University School of Medicine, Chicago,
Author Manuscript
IL 8Department
of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL
Abstract Background—Apamin sensitive small-conductance Ca2+-activated K+ (SK) channels are gated by intracellular Ca2+ through a constitutive interaction with calmodulin. Objective—We hypothesize that arrhythmogenic human calmodulin mutations impede activation of SK channels.
Author Manuscript
Methods—We studied 5 previously published calmodulin mutations (N54I, N98S, D96V, D130G and F90L). Plasmids encoding either wild type (WT) or mutant calmodulin were transiently transfected into human embryonic kidney (HEK) 293 cells that stably express SK2 channels (SK2 Cells). Whole-cell voltage-clamp recording was used to determine apaminsensitive current (IKAS) densities. We also performed optical mapping studies in normal murine
Corresponding Author: Peng-Sheng Chen, MD, 1800 N. Capitol Ave, Room E475, Indianapolis, IN 46202-1228 Telephone: (317) 274-0909, Fax: (317) 962-0588, [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Disclosures: Dr Peng-Sheng Chen has equity interest in Arrhythmotech, LLC. Our laboratory receives equipment donations from Medtronic, St Jude and Cyberonics Inc. Dr. George receives research grants from Gilead Sciences, Inc. and Merck, Inc.
Yu et al.
Page 2
Author Manuscript
hearts to determine the effects of apamin in hearts with (N=7) or without (N=3) pretreatment with sea anemone toxin (ATX II). Results—SK2 cells transfected with WT calmodulin exhibited IKAS density (in pA/pF) of 33.6 [31.4;36.5] (median and confidence interval 25%-75%), significantly higher than that observed for cells transfected with N54I (17.0 [14.0;27.7], p=0.016), F90L (22.6 [20.3;24.3], p=0.011), D96V (13.0 [10.9;15.8], p=0.003), N98S (13.7 [8.8;20.4], p=0.005) and D130G (17.6 [13.8;24.6], p=0.003). The reduction of SK2 current was not associated with a decrease in membrane protein expression or intracellular distribution of the channel protein. Apamin increased the ventricular APD80 (from 79.6 ms [63.4-93.3] to 121.8 ms [97.9-127.2], p=0.010) in hearts pre-treated with ATX-II but not in control hearts. Conclusion—Human arrhythmogenic calmodulin mutations impede the activation of SK2 channels in HEK 293 cells.
Author Manuscript
Keywords Long QT syndrome; catecholaminergic polymorphic ventricular tachycardia; ion channels; cardiac electrophysiology; arrhythmias
Introduction
Author Manuscript Author Manuscript
Small conductance calcium-activated potassium (SK) channels carry a repolarization current responsible for afterhyperpolarization of neurons in the nervous system and contributes to the modulation of intrinsic excitability, synaptic transmission and long-term changes that affect learning and memory formation.1, 2 SK also contributes significantly to the repolarization of atrial myocytes3, 4 and promotes automaticity and atrioventricular node conduction.5 SK is upregulated in atrial fibrillation, heart failure and myocardial infarction.4, 6-8 While SK current is difficult to detect in normal ventricular myocytes at normal pacing rates,3, 6, 9 it is important for ventricular repolarization during bradycardia and atrioventricular (AV) block.10 Apamin is a specific SK current blocker in both neuronal type and cardiac type ion channels.1, 11 SK current blockade by apamin can cause premature ventricular contraction, QT prolongation and torsades de pointes ventricular arrhythmia in normal rabbit ventricles.10 However, there are no reported cases of SK channel mutations in humans with cardiac arrhythmias. Therefore, the importance of SK current in human arrhythmogenesis remains unclear. Calmodulin mutations were reported to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT; N54I, N98S),12 congenital long QT syndrome (LQTS) with recurrent cardiac arrest (D96V, D130G and F142L)13 and childhood and adolescent onset idiopathic ventricular fibrillation (VF) (F90L).14 Because SK channels are gated by intracellular Ca2+ through a constitutive interaction between the pore-forming subunits and calmodulin,1 it is possible that mutant calmodulin may impede the activation of the SK currents. We used human embryonic kidney (HEK) 293 cells that stably express subtype 2 of SK protein (SK2 cells) to test this hypothesis.
Heart Rhythm. Author manuscript; available in PMC 2017 August 01.
Yu et al.
Page 3
Author Manuscript
Methods The study was approved by the Institutional Biosafety Committee and Institutional Animal Care and Use Committee of the Indiana University and the Methodist Research Institute, Indianapolis, Indiana. Detailed methods are included in an online supplement. Transfection of wild type and mutant calmodulins SK2 cells were transfected with various plasmids. The amount of wild type calmodulin plasmids (WT-CaM/pIRES2-EGFP or WT-CaM/pIRES2-dsRED) or mutant calmodulin plasmids (N54I, F90L, D96V, N98S, D130G/pIRES2-EGFP) transfected was either 1 µg each or 1 µg total in combination (for co-expression experiments). Patch-clamp experiments
Author Manuscript
Whole cell voltage-clamp technique was performed.11 The Tyrode’s (bath) solution contains (in mM) NaCl 140, KCl 5.4, MgCl2 1.2, HEPES 5, NaH2PO4 0.33, CaCl2 1.8 and D-glucose 10 (pH 7.4 adjusted with NaOH). The pipette solution contained (in mM) K-Gluconate 144, MgCl2 1.15, EGTA 1, HEPES 10 and different concentration of CaCl2 (pH 7.2 adjusted with KOH). Free Ca2+ concentration was 1.0 μmol/L for all experiments except when testing calcium sensitivities (0.1 to 10 μM). All experiments were carried out at 37°C. SK2 current density was calculated as the difference of current at 0 mV before and after exposure to apamin (500 nM). Immunofluorescence imaging
Author Manuscript
HEK 293 cells and SK2 cells were seeded and grown on coated glass coverslips. The cells were stained with anti-SK2 rabbit polyclonal antibody, incubated with Alexa Fluor 568 goat anti-rabbit IgG secondary and counterstained with DAPI. The cover glasses were then mounted onto a glass slide and imaged by a Leica TCS SP8 laser scanning confocal microscope. To minimize variability, we plated cells on the same day, introduced plasmids in the same way and immunostained all samples altogether. We used the same imaging conditions and acquired images within 2~3 hours to reduce photobleaching. We then compared cells side by side in the same region in the same sample. Western blot analysis
Author Manuscript
HEK 293 cells and SK2 cells were washed in cold PBS and subsequently lysed and homogenized in 10 mM cold sodium bicarbonate. The membrane fractions were harvested by centrifugation. The protein concentrations were measured with Lowry protein assay. Sixty µg of microsomes were subjected to a SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The blot was probed with a rabbit anti-SK2 polyclonal antibody. Antibody-bound protein bands were visualized with 125I-protein A followed by autoradiography. The membrane was then scanned in a flat-bed scanner at 600 dpi and the staining densities were analyzed using ImageJ 1.49b.
Heart Rhythm. Author manuscript; available in PMC 2017 August 01.
Yu et al.
Page 4
Optical mapping studies
Author Manuscript
We used 10 wild type (WT) mice for optical mapping of membrane potential. The hearts were Langendorff perfused with 37°C oxygenated Tyrode’s solution and stained with dye di-4-ANEPPS. Blebbistatin (10 mM) was added to the Tyrode’s solution. Hearts were illuminated with a solid-state laser. Seven hearts were first treated with 15 nM sea anemone toxin (ATX-II) to prolong QT interval, followed by 100 nM of apamin. The remaining 3 hearts were treated with 100 nM of apamin only. The action potential duration measured to 80% repolarization (APD80) was determined at pacing cycle lengths (PCL) of 200 ms. Statistical analysis
Author Manuscript
Data in text and figures are presented as median and confidence interval [25th percentile; 75th percentile] except the figure of normalized calcium sensitivity curve which is presented as mean ± standard error. Nonparametric tests were used for data analyses. Mann-Whitney U test was conducted to compare SK2 current densities between groups with or without transfected calmodulins. Kruskal-Wallis test was performed to test the existence of a statistically significant difference among mutant calmodulins. Repeated measures analyses of variance (RMANOVA) was used to compare the effects of WT:D96V ratio on current density and APD changes in the optical mapping studies. A p value ≤ 0.05 was considered statistically significant. Statistical analyses were performed using SPSS (IBM, Chicago, IL, USA, version 21).
Results
Author Manuscript
Figure 1A illustrates representative current tracings from an SK2 cell before (trace a) and after (trace b) apamin, respectively, obtained by the descending voltage ramp protocol shown in the inset. Trace b shows the small endogenous current (
